Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage

ABSTRACT

The present invention is directed stromal cell derived factor-1 peptides that have been mutated to make them resistant to digestion by the proteases dipeptidyl peptidase IV (DPPIV) and matrix metalloproteinase-2 (MMP-2) but which maintain the ability of native SDF-1 to attract T cells. The mutants may be attached to membranes formed by self-assembling peptides and then implanted at sites of tissue damage to help promote repair.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. 120, 121, or 365(c)of U.S. patent application Ser. No. 11/976,032 filed Oct. 19, 2007,which claims priority to and benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Nos. 60/929,353 filed on Jun. 22, 2007 and60/853,441 filed on Oct. 23, 2006. The contents of each of theseapplications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to stromal cell derived factor-1(SDF-1) peptides that have been mutated in a manner that preserves theirability to attract cells but which makes them resistant to inactivationby proteases, particularly matrix metalloproteinase-2 (MMP-2) and/ordipeptidyl peptidase IV (DPPIV/CD26). When delivered to damaged tissue,these mutants promote tissue repair. The peptides should also be usefulin the treatment of many conditions, including ulcers in thegastrointestinal tract or elsewhere, wounds resulting from accident,surgery or disease; and cardiac tissue damaged as the result of amyocardial infarction. The peptides should also be useful in treatingdiabetic patients to make them less susceptible to damage caused bywounds, ulcers or lesions. In an especially preferred embodiment, themutated forms of SDF-1 are delivered to damaged tissue using a membraneformed by self-assembling peptides.

BACKGROUND OF THE INVENTION

Stromal cell derived factor-1 (SDF-1, or CXCL12) is a 68 amino acidmember of the chemokine family which attracts resting T-lymphocytes,monocytes and CD34+ stem cells. It is commonly found in two differentforms SDF-1α and SDF-1β which are the result of differential mRNAsplicing (U.S. Pat. No. 5,563,048). These forms are essentially the sameexcept that SDF-1β is extended by four amino acids (-Arg-Phe-Lys-Met) atthe C terminus. Both forms of SDF-1 are initially made with a signalpeptide, 21 amino acids in length, that is cleaved to make the activepeptide (U.S. Pat. No. 5,563,048). For the purposes of the presentinvention, it will be understood that the term “SDF-1” refers to theactive form of the peptide, i.e., after cleavage of the signal peptide,and encompasses both SDF-1α and SDF-1β.

It has also been shown that the full length, 68 amino acid, SDF-1sequence is not needed for activity. Peptides that have at least thefirst eight N-terminal residues of SDF-1 maintain the receptor bindingand bioactivity of the full peptide, albeit at a reduced potency. Forexample, SDF-1, 1-8, 1-9, 1-9 dimer, and 1-17 induce intracellularcalcium and chemotaxis in T lymphocytes and CEM cells and bind to CXCchemokine receptor 4 (CXCR4). However, native SDF-1 has half-maximalchemoattractant activity at 5 nM, whereas the 1-9 dimer requires 500 nMand is therefore 100-fold less potent. The 1-17 and a 1-9 monomeranalogs are 400- and 3600-fold, respectively, less potent than SDF-1.SDF-1 variants with C-terminal cyclization have been described that havea higher CXCR4 receptor binding affinity and cyclization of this typemay, if desired, be used in connection with the peptides describedherein. For the purposes of the present invention, the term SDF-1 willinclude forms of the peptide that have been truncated at the C terminalend but which maintain SDF-1 biological activity, i.e., which arechemotactic for T lymphocytes and CEM cells and which bind to CXCchemokine receptor 4 (CXCR4). At a minimum, these truncated formsinclude the first eight amino acids at the N-terminal end of thepeptide.

SDF-1 plays a key-role in the homing of hematopoietic stem cells to bonemarrow during embryonic development (Nagasawa, et al., Nature382:635-638 (1996); Zou, et al., Nature 393:595-599 (1998)) and afterstem cell transplantation (Lapidot, et al., Blood 106:1901-1910 (2005)).In addition to its role in stem cell homing, SDF-1 is also important incardiogenesis and vasculogenesis. SDF-1 deficient mice die perinatallyand have defects in cardiac ventricular septal formation, bone marrowhematopoiesis and organ-specific vasculogenesis (Nagasawa, et al.,Nature 382:635-638 (1996); Zou, et al., Nature 393:595-599 (1998)). Ithas also been reported that abnormally low levels of SDF-1 are at leastpartially responsible for the impaired wound healing associated withdiabetic patients and that impairment can be reversed by theadministration of this cytokine at the site of tissue damage (Gallagher,et al., J. Clin. Invest. 117:1249-1259 (2007)).

In the normal adult heart, SDF-1 is expressed constitutively, butexpression is upregulated within days after myocardial infarction(Pillarisetti, et al., Inflammation 25:293-300 (2001)). Askari et al.increased SDF-1 expression 8 weeks after myocardial infarction byintramyocardial transplantation of stably transfected cardiacfibroblasts overexpressing SDF-1 in combination with G-CSF therapy(Lancet 362:697-703 (2003)). This was associated with higher numbers ofbone marrow stem cells (c-Kit or CD34 positive) and endothelial cells inthe heart and resulted in an increase of vascular density and animprovement of left ventricular function. These studies suggest that theinsufficiency of the naturally-occurring myocardial repair process maybe in part due to inadequate SDF-1 availability. Hence, the delivery ofSDF-1 in a controlled manner after myocardial infarction may attractmore progenitor cells and thereby promote tissue repair (Penn, et al.,Int. J. Cardiol. 95(Suppl. 1):S23-S25 (2004)). Apart from this, theadministration of SDF-1 may be used to improve the healing of wounds orulcers in patients, especially those with diabetes.

One way that may be used for the sustained delivery of drugs at a siteof tissue damage is through the use of biologically compatiblemembranes. Certain peptides are capable of self-assembly when incubatedin the presence of a low concentration of monovalent metal cation (U.S.Pat. No. 5,670,483; U.S. Pat. No. 6,548,630). Assembly results in theformation of a gel-like membrane that is non-toxic, non-immunogenic andrelatively stable to proteases. Once formed, membranes are stable inserum, aqueous solutions and cell culture medium. They can be made understerile conditions, are capable of supporting the growth of cells andare slowly digested when implanted in an animal's body. Thesecharacteristics make the membranes well suited as devices for thedelivery of therapeutic agents (US 20060148703 and 20060088510).

SUMMARY OF THE INVENTION

The present invention is based, in part, on experiments that had astheir hypothesis that the beneficial effect of stromal cell derivedfactor-1 (SDF-1) in the recovery of damaged cardiac tissue is limited byhigh concentrations of the protease matrix metalloproteinase-2 (MMP-2)present in such tissue. More specifically, it was proposed that theMMP-2 cleaves SDF-1 and thereby eliminates its ability to attractprogenitor cells to the site of tissue damage.

In order to test this hypothesis, the inventors developed mutated formsof SDF-1 that retain their ability to attract T cells but which areresistant to MMP-2 digestion. The mSDF-1 peptides were attached to aspecially designed membrane formed by self-assembling peptides and thentested in an animal model of cardiac damage. It was found that mSDF-1attached to membranes and implanted into the myocardium of test animalsimproved cardiac recovery to a greater extent than either SDF-1 ormSDF-1 that was not attached to membranes.

In addition, the inventors found that truncated forms of SDF-1 maintainbioactivity and, as with the full length peptide, mutations in thefourth or fifth amino acids protect the peptide from protease digestion.

In its first aspect, the invention is directed to mutant forms of SDF-1(mSDF-1) which are characterized by a change in the fourth and/or thefifth amino acid from the N-terminus of unmutated SDF-1(KPVSLSYRCPCRFFESHVARANVKHLKI LNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK(SEQ ID NO:52)). Thus, the fourth amino acid is changed to an amino acidother than S and/or the fifth amino acid is changed to an amino acidother than L. As discussed above, truncated forms of the full lengthSDF-1 peptide maintain biological activity provided that the first eightamino acids (highlighted in the sequence shown above) are present andthese truncated forms may also be made protease resistant by mutatingthe fourth and/or fifth position. The invention includes thesebiologically active truncated mutants as well. Put another way, theinvention includes peptides comprising the amino acid sequence of atleast amino acids 1-8 of SEQ ID NO:52, which are optionally extended atthe C terminus by all or any portion of the remaining sequence of SEQ IDNO:52, shown as amino acids 9-68. In all cases, the peptide will have asequence corresponding to that given in SEQ ID NO:52 except that therewill be a proteinogenic amino acid other than S at position 4 and/or aproteinogenic amino other than L at position 5.

For the purposes of the present invention, all peptide sequences arewritten from the N terminus (far left) to the C terminus (far right) andunless otherwise indicated, all amino acids are “proteinogenic” aminoacids, i.e., they are the L-isomers of: alanine (A); arginine (R);asparagine (N); aspartic acid (D); cysteine (C); glutamic acid (E);glutamine (Q); glycine (G); histidine (H); isoleucine (I); leucine (L);lysine (K); methionine (M); phenylalanine (F); proline (P); serine (S);threonine (T); tryptophan (W); tyrosine (Y); or valine (V). Mutant SDF-1peptides may be abbreviated herein as “mSDF-1,” “mSDF” or SDF(NqN')where N is the one letter designation of the amino acid originallypresent, q is its position from the N terminus of the peptide and N′ isthe amino acid that has replaced N. It will also be understood that,although SEQ ID NO:52 shows the intact full length sequence of SDF-1α,this sequence may be extended at the C terminus by up to four more aminoacids, in particular with the sequence—R-F-K-M. Thus, the inventionincludes mutant forms of both SDF-1α and SDF-1β (see U.S. Pat. No.5,563,048). In some instances, peptides that have been mutated by theaddition of amino acids at the N terminus are abbreviated as “Xp-R”where X is a proteinogenic amino acid, p is an integer and R is thepeptide prior to extension. It will also be understood that, unlessotherwise indicated, all pharmaceutically acceptable forms of peptidesmay be used, including all pharmaceutically acceptable salts.

The mSDF-1 peptides must maintain chemoattractant activity with asensitivity (as determined by, e.g., the effective concentration neededto obtain 50% of maximal response in the assays of Jurkat T cellmigration described herein) of at least 1/10 the sensitivity ofunmutated SDF-1. In addition, the mSDF-1 peptides must be resistant toloss of this chemoattractant activity due to cleavage by matrixmetalloproteinase-2 (MMP-2). Preferably the rate of inactivation ofmSDF-1 is less than ½ (and more preferably, less than ¼ or 1/10) therate of inactivation of SDF-1.

In one embodiment, the mSDF-1 peptide has the sequence: KPVXLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCI DPKLKWIQEYLEKALNK (SEQ IDNO:53) where X is any of the 20 proteinogenic amino acids except S. Themost preferred of these peptides is SDF(S4V) which has the sequence:KPVVLSYRCPCRFFESHVARANVKHLKILNTPNC ALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK(SEQ ID NO:54). SEQ ID 53 and 54 show the full sequence of SDF-1peptides. However, it will be understood that truncated versions of thepeptides will maintain activity as long as the first eight N-terminusamino acids are present. These are also part of the invention and may bemade protease resistant by mutating the amino acids at positions 4and/or 5.

In another embodiment, the mSDF-1 peptide has the sequence: KPVSXSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQV CIDPKLKWIQEYLEKALNK (SEQ IDNO:55) where X is any of the 20 proteinogenic amino acids except L, W orE. The most preferred of these peptides is SDF(L5P) which has thesequence: KPVSPSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK (SEQ ID NO:56). Again, peptidesthat are truncated and which have at least the first eight amino acidsof SEQ ID NO:55 or 56 are included in the invention. They may beextended at the C terminus by additional amino acids from the sequencesshown above.

The longest mSDF-1 peptides presented above are 68 amino acids inlength. However, unless otherwise indicated, it will also be understoodthat one additional proteinogenic amino acid may be added to the Nterminus without substantially changing chemoattractant activity orMMP-2 resistance. Moreover, the addition of an amino acid at the Nterminus represents a preferred embodiment since this will have theeffect of making the peptide resistant to digestion by a second commonpeptidase, dipeptidyl peptidase IV (DPPIV/CD26, abbreviated herein as“DPPIV”).

DPPIV is a 110-kD glycoprotein which is expressed in renal proximaltubules, in intestinal epithelial cells, liver, placenta and lung andwhich cleaves peptides that have a proline in the second position fromthe N terminus (Kikawa, et al., Biochim. Biophys. Acta 1751:45-51(2005)). SDF-1 has a proline in the second position (as can be seenabove in SEQ ID NO:52) and is therefore cleaved by DPPIV between thisproline and the following valine (Narducci, et al., Blood 107:1108-1115(2006); Christopherson, Exp. Hematol. 34:1060-1068 (2006)).

One way to eliminate the proteolytic effect of DPPIV would be to changethe proline in position 2 of SDF-1 (see SEQ ID NO:52). However, thisproline is essential for SDF-1's biological activity and thereforecannot be replaced and maintain a therapeutically effective peptide.However, activity can be maintained and a DPPIV resistant peptide madeby adding one to four amino acids (or an organic group) to the Nterminus of SEQ ID:52. For example, it has been experimentally foundthat resistance to DPPIV cleavage can be obtained by adding a serine tothe N terminus of the peptide.

Thus, in another aspect, the invention is directed to the peptideX_(p)-SDF-1, where X is preferably, any proteinogenic amino acid, p isan integer between 1 and 4, and SDF-1 is as shown in SEQ ID NO:52. Inpreferred embodiments, n=1. It will be understood that when p is greaterthan 1, each of the 2-4 added amino acids may independently be chosenfrom any of the proteinogenic amino acids described herein, i.e., any ofthese proteinogenic amino acids may be in the first position, any in thesecond position, etc.

SDF-1 may also be made resistant to DPPIV by adding a “proteaseprotective organic group” to the N-terminus. A “protease protectiveorganic group” is defined herein as an organic group, other than aproteinogenic amino acid, that, when added to the N terminal amino acidof SDF-1, results in a modified peptide that maintains at least 10% (andpreferably at least 50% or 80%) of the chemoattractant activity ofunmodified SDF-1(as determined by, e.g., assays of Jurkat T cellmigration described herein) and which, in addition, is inactivated byDPPIV at a rate of less than 50% (and more preferably, at a rate of lessthan 25% or 10%) the rate of inactivation of unmodified SDF-1. Forexample, X may be: R¹—(CH₂)_(d)—, where d is an integer from 0-3, and R¹is selected from: hydrogen (with the caveat that when R¹ is hydrogen, dmust be at least 1); a branched or straight C₁-C₃ alkyl; a straight orbranched C₂-C₃ alkenyl; a halogen, CF₃; —CONR⁵R⁴; —COOR⁵; —COR⁵;—(CH₂)_(q)NR⁵R⁴; —(CH₂),_(q)SOR⁵; —(CH₂)_(q)SOR⁵, —(CH₂)_(q)SO₂NR⁵R⁴;and OR⁵, where R⁴ and R⁵ are each independently hydrogen or a straightor branched C₁-C₃ alkyl. In instances where an organic group is used forX, p should be 1. In addition, X may represent a proteinogenic aminoacid as discussed above, so that 1-4 amino acids are added to SDF-1, andone or more of these added amino acids may be substituted with aprotease protective organic group.

In the formula X_(p)-SDF-1, SDF-1 may optionally include any of themutations in positions 4 and/or 5 of SEQ ID NO:52 as described above.Thus, the invention encompasses peptides of the form X_(p)-mSDF-1, whereX and p are as defined above and mSDF-1 is selected from: SEQ ID NO:53;SEQ ID NO:54; SEQ ID NO:55; and SEQ ID NO:56. These doubly mutatedpeptides will be resistant to both DPPIV and MMP-2.

The invention also encompasses fusion proteins in which any of the abovemSDF-1, X_(p)-SDF-1 or X_(p)-mSDF-1 sequences are linked toself-assembling peptides capable of forming a biologically compatiblemembrane. Membranes with attached protease resistant SDF-1 can beimplanted in a patient at a site of tissue damage, especially cardiactissue damage, wounds (whether accidental, surgical or the result ofdisease) or ulcers and will maintain the SDF-1 biological activity atthat site for a prolonged period of time. Fusion proteins are formedeither by joining the C terminal end of a protease resistant SDF-1peptide directly to the N terminal end of a self-assembling peptide orthe two peptides can be joined by a linker sequence. Thus, the inventionincludes fusion proteins of the formula: A-(L)_(n)-(R)_(q), where n isan integer from 0-3, q is an integer from 1-3, A is one of the proteaseresistant SDF-1 peptides (i.e., mSDF-1, X_(p)-SDF-1 or X_(p)-mSDF-1)described above, L is a linker sequence 3-9 amino acids long, and R is aself-assembling peptide selected from the group consisting of:

AKAKAEAEAKAKAEAE,; (SEQ ID NO: 1) AKAEAKAEAKAEAKAE,; (SEQ ID NO: 2)EAKAEAKAEAKAEAKA,; (SEQ ID NO: 3) KAEAKAEAKAEAKAEA,; (SEQ ID NO: 4)AEAKAEAKAEAKAEAK,; (SEQ ID NO: 5) ADADARARADADARAR,; (SEQ ID NO: 6)ARADARADARADARAD,; (SEQ ID NO: 7) DARADARADARADARA,; (SEQ ID NO: 8)RADARADARADARADA,; (SEQ ID NO: 9) ADARADARADARADAR,; (SEQ ID NO: 10)ARADAKAEARADAKAE,; (SEQ ID NO: 11) AKAEARADAKAEARAD,; (SEQ ID NO: 12)ARAKADAEARAKADAE,; (SEQ ID NO: 13) AKARAEADAKARADAE,; (SEQ ID NO: 14)AQAQAQAQAQAQAQAQ,; (SEQ ID NO: 15) VQVQVQVQVQVQVQVQ,; (SEQ ID NO: 16)YQYQYQYQYQYQYQYQ,; (SEQ ID NO: 17) HQHQHQHQHQHQHQHQ,; (SEQ ID NO: 18)ANANANANANANANAN,; (SEQ ID NO: 19) VNVNVNVNVNVNVNVN,; (SEQ ID NO: 20)YNYNYNYNYNYNYNYN,; (SEQ ID NO: 21) HNHNHNHNHNHNHNHN,; (SEQ ID NO: 22)ANAQANAQANAQANAQ,; (SEQ ID NO: 23) AQANAQANAQANAQAN,; (SEQ ID NO: 24)VNVQVNVQVNVQVNVQ,; (SEQ ID NO: 25) VQVNVQVNVQVNVQVN,; (SEQ ID NO: 26)YNYQYNYQYNYQYNYQ,; (SEQ ID NO: 27) YQYNYQYNYQYNYQYN,; (SEQ ID NO: 28)HNHQHNHQHNHQHNHQ,; (SEQ ID NO: 29) HQHNHQHNHQHNHQHN,; (SEQ ID NO: 30)AKAQADAKAQADAKAQAD,; (SEQ ID NO: 31) VKVQVDVKVQVDVKVQVD,; (SEQ ID NO:32) YKYQYDYKYQYDYKYQYD,; (SEQ ID NO: 33) HKHQHDHKHQHDHKHQHD,; (SEQ IDNO: 34) RARADADARARADADA,; (SEQ ID NO: 35) RADARGDARADARGDA,; (SEQ IDNO: 36) RAEARAEARAEARAEA,; (SEQ ID NO: 37) KADAKADAKADAKADA,; (SEQ IDNO: 38) AEAEAHAHAEAEAHAH,; (SEQ ID NO: 39) FEFEFKFKFEFEFKFK,; (SEQ IDNO: 40) LELELKLKLELELKLK,; (SEQ ID NO: 41) AEAEAKAKAEAEAKAK,; (SEQ IDNO: 42) AEAEAEAEAKAK,; (SEQ ID NO: 43) KAKAKAKAEAEAEAEA,; (SEQ ID NO:44) AEAEAEAEAKAKAKAK,; (SEQ ID NO: 45) RARARARADADADADA,; (SEQ ID NO:46) ADADADADARARARAR,; (SEQ ID NO: 47) DADADADARARARARA,; (SEQ ID NO:48) HEHEHKHKHEHEHKHK,; (SEQ ID NO: 49) VEVEVEVEVEVEVEVEVEVE,; (SEQ IDNO: 50) and RFRFRFRFRFRFRFRFRFRF,. (SEQ ID NO: 51)

The most preferred self-assembling peptide is: RARADADARARADADA, (SEQ IDNO:35) with q=1; and preferred protease resistant SDF-1 peptides areSDF(S4V) and X_(p)-SDF(S4V), especially where p=1. When joined together,the resulting fusion proteins are, for convenience, abbreviated asSDF(S4V)-RAD or X_(p)-SDF(S4V)-RAD. Preferred linker sequences occurwhen n=1 and L is GGGGGG (abbreviated as “6G,” SEQ ID NO:57); GIVGPL(SEQ ID NO:58) and PVGLIG (SEQ ID NO:59). The lattermost represents anMMP-2 cleavage site (“MCS”). GIVGPL (SEQ ID NO:58) represents ascrambled version of MCS and is abbreviated as “SCR.” Surprisingly, thissequence was also found to undergo MMP-2 cleavage, although at a slowerrate than MCS. Preferred, fusion proteins containing linker sequencesare: SDF(S4V)-6G-RAD; X_(p)-SDF(S4V)-6G-RAD; SDF(S4V)-MCS-RAD;X_(p)-SDF(S4V)-MCS-RAD; SDF(S4V)-SCR-RAD; and XP- SDF(S4V)-SCR-RAD.Again, p is preferably 1.

In another aspect, the invention is directed to nucleic acids comprisinga nucleotide sequence encoding any of the protease resistant peptides orfusion proteins described above, vectors in which these nucleic acidsare operably linked to a promoter sequence and host cells transformedwith the vectors. The term “operably linked” refers to genetic elementsthat are joined in a manner that enables them to carry out their normalfunctions. For example, a sequence encoding a peptide is operably linkedto a promoter when its transcription is under the control of thepromoter and the transcript produced is correctly translated into thepeptide.

Preferred nucleic acids encoding protease resistant SDF-1 peptides andfusion proteins include:

(SEQ ID NO: 60)aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaag; (SEQ ID NO:61)aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagtgaggaatcgtgggacctctgcgtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc; (SEQ ID NO: 62)aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagcctgtgggactgatcggagtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc; and (SEQ ID NO: 63)aagcccgtcgtcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagggaggcgggggaggtgggcgtgcccgtgccgacgccgacgcccgtgcccgtgccgacgccgacgcc;

In another aspect, the invention is directed to a biologicallycompatible membrane formed from self-assembling peptides as described inpublished US applications 20060148703 and 20060088510 which have mSDF-1,Xp-SDF-1 or Xp-mSDF-1 peptides attached. The term “biologicallycompatible” indicates that the membranes are non-toxic and can beimplanted in a patient without triggering an immune response. Between0.1% and 10% (and preferably 0.5-5%) of the peptides that assemble intothe membrane are bound to a mutant SDF-1. Binding may be either covalentor noncovalent. Noncovalent bonding occurs when protease resistant SDF-1peptides are simply trapped in the membrane matrix and when proteaseresistant SDF-1 peptides are bound to self-assembling peptides in themembrane by biotin/avidin linkages. As used herein, the term “avidin” isintended to include streptavidin as well. The membranes may, optionally,have other therapeutic agents, e.g., PDGF or interleukin-8, attached aswell.

The use of biotin and avidin for linking molecules is well known in theart and standard methodology can be used for attaching proteaseresistant SDF-1 peptides to self-assembling peptides either before orafter membrane formation. Specific methodology for using biotin/avidinin connection with self-assembling membranes has been described in US20060088510 and this methodology can be applied to forming membraneswith attached cytokine. In order to prevent steric interference betweenthe biotin/avidin groups and protease resistant peptides, a spacer maybe included between the two. The spacer can take the form of 1-15(preferably 1-10) fatty acids or 1-15 (preferably)-10) amino acids andshould separate the protease resistant SDF-1 peptide from theself-assembling peptide by at least an additional 12 angstroms and by nomore than an additional 250 angstroms. Methodology for incorporatingspacers of this type is well known in the art. In a preferredembodiment, about 1% of the self-assembling peptides used in membranesare attached to protease resistant SDF-1. It is also preferable that theself-assembling peptides making up membranes be homogeneous, i.e., thatall of the peptides are identical.

As an alternative, protease resistant SDF-1 peptides may be joined to aself-assembling peptide that is part of the membrane by a peptide bond,i.e., the protease resistant SDF-1 may be part of a fusion protein inwhich it is joined to a self-assembling peptide either directly or viaan intervening linker amino acid sequence. Any of the fusion proteinsdescribed above may be used, with SDF(S4V)-6G-RAD;X_(p)-SDF(S4V)-6G-RAD; SDF(S4V)-MCS-RAD; X_(p)-SDF(S4V)-MCS-RAD;SDF(S4V)-SCR-RAD and X_(p)- SDF(S4V)-SCR-RAD being particularlypreferred. The membranes are made from the fusion proteins (or from theself-assembling peptides) by taking advantage of the fact that theself-assembling peptides described herein do not congregate together inwater, but assemble into a membrane in the presence of a lowconcentration of monovalent metal cation. Thus, for example, fusionproteins may be made under conditions in which self-assembly does notoccur and then exposed to conditions that promote membrane formation,e.g., low monovalent metal cation concentration. The end result is amatrix which can be implanted into a patient and which will maintain ahigh concentration of SDF-1 biological activity at the site ofimplantation. Alternatively, the fusion proteins can be incorporatedinto an injectable pharmaceutical composition at a concentration ofmonovalent cation that is too low to induce self-assembly and can thenadministered to a patient to induce membrane formation in vivo.

The mutated SDF-1 peptides are resistant to cleavage by MMP-2 and/orDPPIV but maintain at least a portion (at least 10% and preferably morethan 25%, 50% or 80%) of the chemoattractant activity of native SDF-1.Thus, they are ideally suited for use at sites, such as damaged cardiactissue, where MMP-2 (or DPPIV) is present at a high concentration. Inaddition, an MMP-2 cleavage site can, if desired, be placed in linkerregions joining the SDF-1 peptides to the self-assembling peptides. Thiswill allow for the protease resistant SDF-1 peptides to be released froman implanted membrane over time.

The compositions described above should be useful in the treatment ofany disease or condition characterized by high concentrations of MMP-2and/or DPPIV where attraction of stem cells might induce regeneration orhealing. This would include the treatment of inflammatory and ischemicdiseases such as stroke, limb ischemia; wound healing: and diabeticulcers. In an especially preferred embodiment, the invention is directedto a method of treating damaged cardiac tissue, for example subsequentto a heart attack, by injecting or implanting any of the biologicallycompatible peptide membranes or fusion proteins described above at ornear the site of damage. Preferably, membranes will be injected orimplanted directly into the damaged tissue, e.g., myocardium, of apatient. The membranes should be large enough to prevent the proteaseresistant SDF-1 from being washed away by bodily fluids and a sufficientamount of mSDF-1 should be present to promote the migration of T cellsto the site of injury. Guidance with regard to these parameters isprovided by the experiments described herein.

DESCRIPTION OF THE INVENTION

The present invention is based upon the concept that the recovery ofdamaged tissue, e.g., damaged cardiac tissue, is promoted by exposingthe tissue to SDF-1 that has been mutated to make it resistant to MMP-2and/or DPPIV cleavage and which is delivered by means of a membraneformed by spontaneously assembling peptides. The self-assemblingpeptides have been described in U.S. Pat. Nos. 5,670,483 and 6,548,630(hereby incorporated by reference in their entirety). Methods ofattaching factors to membranes and the use of the membranes indelivering therapeutic agents to cardiac tissue have also been described(see published US applications 20060148703 and 20060088510, herebyincorporated by reference in their entirety). The same procedures formaking and using membranes may be applied to the present invention.

Description of Self-Assembling Peptides

The peptides used for self-assembly should be at least 12 residues inlength and contain alternating hydrophobic and hydrophilic amino acids.Peptides longer than about 200 amino acids tend to present problems withrespect to solubility and membrane stability and should therefore beavoided. Ideally, peptides should be about 12-24 amino acids in length.

The self-assembling peptides must be complementary. This means that theamino acids on one peptide must be capable of forming ionic bonds orhydrogen bonds with the amino acids on another peptide. Ionic bondswould form between acidic and basic amino acid side chains. Thehydrophilic basic amino acids include Lys, Arg, His, and Orn. Thehydrophilic acidic amino acids are Glu and Asp. Ionic bonds would formbetween an acidic residue on one peptide and a basic residue on another.Amino acids that form hydrogen bonds are Asn and Gln. Hydrophobic aminoacids that may be incorporated into peptides include Ala, Val, Ile, Met,Phe, Tyr, Trp, Ser, Thr, and Gly.

Self-assembling peptides must also be “structurally compatible.” Thismeans that they must maintain an essentially constant distance betweenone another when they bind. Interpeptide distance can be calculated foreach ionized or hydrogen bonding pair by taking the sum of the number ofunbranched atoms on the side-chains of each amino acid in the pair. Forexample, lysine has five and glutamic acid has four unbranched atoms ontheir side chains. An interaction between these two residues ondifferent peptides would result in an interpeptide distance of nineatoms. In a peptide containing only repeating units of EAK, all of theion pairs would involve lysine and glutamate and therefore a constantinterpeptide distance would be maintained. Thus, these peptides would bestructurally complementary. Peptides in which the variation ininterpeptide distance varies by more than one atom (about 3-4 angstroms)will not form gels properly. For example, if two bound peptides have ionpairs with a nine-atom spacing and other ion pairs with a seven-atomspacing, the requirement of structural complementarity would not havebeen met. A full discussion of complementarity and structuralcompatibility may be found in U.S. Pat. Nos. 5,670,483 and 6,548,630.

It should also be recognized that membranes may be formed from either ahomogeneous mixture of peptides or a heterogeneous mixture of peptides.The term “homogeneous” in this context means peptides that are identicalwith one another. “Heterogeneous” indicates peptides that bind to oneanother but which are structurally different. Regardless of whetherhomogenous or heterogeneous peptides are used, the requirements withrespect to the arrangement of amino acids, length, complementarity, andstructural compatibility apply. In addition, it should be recognizedthat the carboxyl and amino groups of the terminal residues of peptidescan either be protected or not protected using standard groups.

Making of Peptides

The self-assembling and protease resistant SDF-1 peptides of the presentinvention can be made by solid-phase peptide synthesis using standardN-tert-butyoxycarbonyl (t-Boc) chemistry and cycles usingn-methylpyrolidone chemistry. Once peptides have been synthesized, theycan be purified using procedures such as high pressure liquidchromatography on reverse-phase columns. Purity may also be assessed byHPLC and the presence of a correct composition can be determined byamino acid analysis. A purification procedure suitable for mSDF-1peptides is described in the Examples section.

Fusion proteins may either be chemically synthesized or made usingrecombinant DNA techniques. The full sequences of these proteins aredescribed herein and examples are provided of DNA sequences that can beused in producing them.

Binding of SDF-1 to Self-Assembling Peptides

Several strategies may be used for attaching protease resistant SDF-1 toself-assembling peptides. One strategy is non-covalent binding which haspreviously been shown to be effective in delivering PDGF-BB, a growthfactor, to tissues (Hsieh, et al., J. Clin. Invest. 116:237-248 (2006)).

A second attachment strategy is the biotin-sandwich method (Davis, etal., Proc. Nat'l Acad. Sci. USA 103:8155-8160 (2006)) in which aprotease resistant SDF-1 is biotinylated and bound to biotinylatedpeptides using tetravalent streptavidin as a linker. To accomplish this,the protease resistant SDF-1 may be coupled to the 15 amino acidsequence of an acceptor peptide for biotinylation (referred as AP; Chen,et al., Nat. Methods 2:99-104 (2005)). Because the active site of SDF-1is situated near the amino terminus, fusion proteins should be made byincorporating the extra sequences at the C-terminus. The acceptorpeptide sequence allows site-specific biotinylation by the E. colienzyme biotin ligase (BirA; Chen, et al., Nat. Methods 2:99-104 (2005)).Many commercial kits are available for biotinylating proteins. However,many of these kits biotinylate lysine residues in a nonspecific manner,and this may reduce mSDF-1 activity as it has been shown that theN-terminal lysine of SDF-1 is crucial for receptor binding and activity(Crump, et al, EMBO J. 16:6996-7007 (1997)). Biotinylatedself-assembling peptides are made by MIT Biopolymers laboratory and whenmixed in a 1 to 100 ratio with native self-assembling peptides,self-assembly of nanofibers should not be disturbed (Davis, et al.,Proc. Nat'l Acad. Sci. USA 103:8155-8160 (2006)).

A third targeting strategy is direct incorporation of protease resistantSDF-1 peptides into self-assembling nanofibers by construction of afusion protein of mutated SDF-1 with a self-assembling peptide. Forexample an mSDF-1 may be coupled to the 16 amino acid sequence of SEQ IDNO:35. This “RAD” portion of the fusion protein will incorporate intothe nanofiber scaffold while assembling.

Formation of Membranes

The self-assembling peptides and fusion proteins described herein willnot form membranes in water, but will assemble in the presence of a lowconcentration of monovalent metal cation. The order of effectiveness ofthese cations is Li⁺>Na⁺>K⁺>Cs⁺(U.S. Pat. No. 6,548,630). Aconcentration of monovalent cation of 5 mM should be sufficient forpeptides to assemble and concentrations as high as 5 M should still beeffective. The anion associated with the monovalent cation is notcritical to the invention and can be acetate, chloride, sulfate,phosphate, etc.

The initial concentration of self-assembling peptide will influence thefinal size and thickness of membranes formed. In general, the higher thepeptide concentration, the higher the extent of membrane formation.Formation can take place at peptide concentrations as low as 0.5 mM or 1mg/ml. However, membranes are preferably formed at higher initialpeptide concentrations, e.g., 10 mg/ml, to promote better handlingcharacteristics. Overall, it is generally better to form membranes byadding peptides to a salt solution rather than adding salt to a peptidesolution.

The formation of membranes is relatively unaffected by pH or bytemperature. Nevertheless, pH should be maintained below 12 andtemperatures should generally be in the range of 4-90° C. Divalent metalcations at concentrations equal to or above 100 mM result in impropermembrane formation and should be avoided. Similarly, a concentration ofsodium dodecyl sulfate of 0.1% or higher should be avoided.

Membrane formation may be observed by simple visual inspection and thiscan be aided, if desired, with stains such as Congo Red. The integrityof membranes can also be observed microscopically, with or withoutstain.

Pharmaceutical Compositions and Dosages

Membranes with attached protease resistant SDF-1 peptides or fusionproteins may be incorporated into a pharmaceutical compositioncontaining a carrier such as saline, water, Ringer's solution and otheragents or excipients. The dosage form will generally be designed forimplantation or injection, particularly into cardiac tissue but topicaltreatments will also be useful, e.g., in the treatment of wounds. Alldosage forms may be prepared using methods that are standard in the art(see e.g., Remington's Pharmaceutical Sciences, 16th ed. A. Oslo. ed.,Easton, Pa. (1980)).

It is expected that the skilled practitioner will adjust dosages on acase by case basis using methods well established in clinical medicine.The optimal dosage will be determined by methods known in the art andwill be influenced by factors such as the age of the patient, diseasestate and other clinically relevant factors.

Examples Example 1 Biological Effects and Protease Resistance of SDF-1Mutants SDF-1 Purification and Expression

The DNA sequence of mature SDF-1α may be cloned from human cDNA intopET-Sumo vector and an extra N-terminal serine residue may beincorporated to facilitate cleavage by Sumo protease (yielding an SDF-1form of 69 AA). Fusion proteins may be made by incorporating RAD or APsequences in reverse primers. Sumo-SDF-1 fusion proteins are expressedin Rosetta DE3 E coli and grown to an optical density of 1.5 (600nm) at37° C. Cells are induced with 0.25 mM isopropyl β-D-thiogalactoside for4 h and harvested by centrifugation. As described below, SDF-1α may bepurified by a 3-step procedure; all steps being performed at 21° C.

Cells from a 4-L growth were lysed in 300 ml lysis buffer (6M Guanidine,20 mM phosphate (pH 7.8), 500 mM NaCl) and homogenized. Debris iscollected by centrifugation at 3000 g. The first purification stepconsisted of capture of the poly-histidine tag present in theSUMO-SDF-1α fusion protein with Nickel-NTA. Nickel-NTA resin was washedwith wash buffer (8M Urea, 500 mM NaCl, 20 mM phosphate (pH 6.2)) andthe bound protein was eluted at pH 4. Further purification and oxidativerefolding were performed on a Cation Exchange HPLC column. The samplewas adjusted to binding buffer (8M Urea, 30 mM 2-mercaptoethanol, 1 mMEDTA, 50 mM Tris pH8) and loaded on the HPLC column. Refolding ofSumo-SDF-1 was performed on the column with a 2 h run of refoldingbuffer (50 mM Tris pH8, 75 mM NaCl, 0.1 mM reduced Glutathione and 0.1mM oxidized Glutathione). Sumo-SDF-1 was eluted with a step gradient(0.5 to 1M NaCl) and concentrated. The SUMO-SDF-1 fusion protein wascleaved by Sumo Protease 1 (1U/ 50 μg protein) in 50 mM Tris pH 8.0, 500mM NaCl. The sample was adjusted to 0.1% trifluoroacetic acid (TFA) andloaded on a C18 Reversed Phase HPLC column for the final purificationstep. The column was subjected to a linear gradient from 30 to 40%acetonitrile in 0.1% TFA. The fractions containing SDF-1 werelyophilized and resuspended. Activity of purified SDF-1 was tested bymigration of Jurkat T-lymphocyte cell line.

Modification of SDF-1 Constructs

SDF-1 fusion constructs were modified by insertional mutagenesis withone of three sequences: one sequence is susceptible to MMP-2 cleavage(MMP cleavage site or MCS), another sequence contains the same aminoacids but in a random order (scrambled sequence, or SCR), and the thirdsequence contains 6 glycines as a linker.

Mutations of the MMP Cleavage Sites in Chemokines

SDF-1 is cleaved by MMP-2 in its active site at the N-terminus, leavingan N-terminal tetrapeptide and inactive SDF-1(5-68). Specificmutagenesis of 4 different amino acids was performed in order to renderSDF-1 resistant to MMP-2 cleavage, based on substrate sequences of MMP-2described by Netzel-Arnett et al (Biochemistry 32:6427-6432 (1993)). Thefour different constructs were expressed and purified as described forSDF-1. Of the 4 different mutations, SDF-1(L5W) and SDF-1(L5E) showedminimal activity on T-cell migration. In contrast, SDF-1(S4V) andSDF-1(L5P) showed bioactivity comparable to native SDF-1. BecauseSDF-1(L5P) was more difficult to purify, SDF-1(S4V) was selected forfurther experiments.

Effect of Mutations on Protease Susceptibility and ChemoattractantActivity

The mutated forms of SDF-1 were examined in an assay of migration ofJurkat T cells at a concentration of 100 nM. This assay indicated thatboth SDF-1(S4V) and SDF-1(L5P) retained most of the activity ofunmutated SDF-1 in promoting T cell migration. This activity was greatlyreduced in SDF-1(L5W) mutants and SDF-1(L5E) mutants.

The susceptibility of the peptides to cleavage by MMP-2 was determinedby incubating the mutants with the enzyme for one hour and thenexamining the incubation product by SDS-PAGE. This revealed that, unlikeSDF-1, the mutants did not undergo a positional shift indicative ofcleavage. MMP-2 incubation was also found to reduce the chemoattractantactivity of SDF-1 but not SDF-1(S4V) as shown by a Jurkat T-cellmigration assay. These results suggest that the S4V variant of SDF-1retains chemokine bioactivity but is resistant to activation by MMP-2.

In vivo Data

A blinded and randomized study was performed to evaluate the effect ofdifferent SDF-1 forms on cardiac function after myocardial infarction inrats. Ejection fraction was measured with a Millar catheter system formeasurement of intraventricular pressures and ventricular volumes. BothSDF-1(S4V)-6G-RAD and SDF-1(S4V)-MCS-RAD significantly increased cardiacfunction 4 weeks after myocardial infarction in rats compared to MI onlygroup. This indicates that both MMP-2 resistance (SDF-1(S4V)) andattachment to membranes are necessary for successful cardiac repairtherapy.

Example 2 Experiments with Truncated Forms of SDF-1

3 truncated forms of SDF-1 were synthesized commercially; all includethe first 17 amino acids of native SDF-1. Two variants of SDF-1 17AAwere designed to be more resistant to MMP-2, based on our prior workwith the entire SDF-1 protein:

SDF-1 17AA: KPVSLSYRCPCRFFESH (SEQ ID 64) SDF-1(S4V) 17AA:KPVVLSYRCPCRFFESH (SEQ ID 65) SDF-1(L5P) 17AA: KPVSPSYRCPCRFFESH (SEQ ID66)

Migration experiments were performed with the Jurkat T-lymphocyte cellline. Truncated SDF-1 17AA was 500 times less potent than native SDF-1but maximal migration induced was similar to native SDF-1. Therefore, if500 times higher concentrations were used compared to full-lengthprotein, the same migratory response of T-lymphocytes should beobserved. The mutated SDF-1(S4V) 17AA and SDF-1(L5P) 17AA were threetimes less potent than SDF-1 17AA without mutation. This is a similarshift to that seen between native SDF-1 and SDF-1(S4V).

Cleavage experiments of the peptides with MMP-2 were performed: 2 nmoleof SDF-1 17AA, SDF-1(S4V) 17AA, and SDF-1(L5P) 17AA were incubated withMMP-2 for 1 h at RT. Proteins were run on an SDS-PAGE showing cleavageof SDF-1 17AA, but not of SDF-1(S4V) 17AA or SDF-1(L5P) 17AA. Thus,these truncated proteins may be useful therapeutically, as they arestill bioactive and also MMP-2 resistant.

All references cited herein are fully incorporated by reference. Havingnow fully described the invention, it will be understood by those ofskill in the art that the invention may be practiced within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

1. An isolated mutant form of stromal cell derived factor-1 (SDF-1)peptide comprising the formula of a mutant SDF-1 (mSDF-1) orX_(p)-mSDF-1, wherein said SDF-1 is a peptide comprising the amino acidsequence of at least amino acids 1-8 of SEQ ID NO:52 and which isoptionally extended at the C terminus by all or any portion of theremaining sequence of SEQ ID NO:52, shown as amino acids 9-68, andwherein: a) X is a proteinogenic amino acid or a protease protectiveorganic group; b) p is any integer from 1 to 4; c) mSDF-1 is a mutantform of said SDF-1 comprising a mutation of the fourth, the fifth, orthe fourth and the fifth amino acid from the N terminus of said SDF-1,wherein mSDF-1 has chemoattractant activity for T cells and isinactivated by matrix metalloproteinase-2 (MMP-2) at a rate that is lessthan one-half of the rate at which native SDF-1 peptide is inactivated;and d) X_(p)-mSDF-1 is a mutant form of said SDF-1 comprising a mutationof the fourth, the fifth, or the fourth and the fifth amino acid fromthe N terminus of said SDF-1, wherein X_(p)-mSDF-1 has chemoattractantactivity for T cells, is inactivated by dipeptidyl peptidase IV (DPPIV)at a rate that is less than one-half of the rate at which native SDF-1is inactivated, and is inactivated by MMP-2 at a rate that is less thanone-half of the rate at which native SDF-1 is inactivated.
 2. Theisolated mutant SDF-1 peptide of claim 1, wherein said mSDF-1 peptide orsaid Xp-mSDF-1 peptide has a mutation of serine to valine at the fourthamino acid.
 3. The isolated mutant SDF-1 peptide of claim 1, whereinsaid mSDF-1 peptide or said Xp-mSDF-1 peptide has a mutation of serineto valine at the fourth amino acid and leucine to proline at the fifthamino acid.
 4. The isolated mutant SDF-1 peptide of claim 1, whereinsaid mutation of the fifth amino acid from the N terminus of said SDF-1peptide is not a mutation to a tryptophan or a glutamic acid amino acidresidue.
 5. The isolated mutant SDF-1 peptide of claim 1, wherein saidisolated mutant peptide is an Xp-mSDF-1 peptide and wherein X is aserine and p equals one.
 6. An isolated mSDF-1 peptide comprising theformula of mSDF-1 or X_(p)-mSDF-1, wherein said SDF-1 is a peptidecomprising the amino acid sequence of at least amino acids 1-17 of SEQID NO:52 and which is optionally extended at the C terminus by all orany portion of the remaining sequence of SEQ ID NO:52, shown as aminoacids 18-68, and wherein: a) X is a proteinogenic amino acid or aprotease protective organic group; b) p is any integer from 1 to 4; c)mSDF-1 is a mutant form of said SDF-1 comprising a mutation of thefourth, the fifth, or the fourth and the fifth amino acid from the Nterminus of said SDF-1, wherein mSDF-1 has chemoattractant activity forT cells and is inactivated by MMP-2 at a rate that is less than one-halfof the rate at which native SDF-1 peptide is inactivated; and d)X_(p)-mSDF-1 is a mutant form of said SDF-1 comprising a mutation of thefourth, the fifth, or the fourth and the fifth amino acid from the Nterminus of said SDF-1, wherein X_(p)-mSDF-1 has chemoattractantactivity for T cells, is inactivated by DPPIV at a rate that is lessthan one-half of the rate at which native SDF-1 is inactivated, and isinactivated by MMP-2 at a rate that is less than one-half of the rate atwhich native SDF-1 is inactivated.
 7. The isolated mutant SDF-1 peptideof claim 6,wherein said mSDF-1 peptide or said Xp-mSDF-1 peptide has amutation of serine to valine at the fourth amino acid.
 8. The isolatedmutant SDF-1 peptide of claim 6, wherein said mSDF-1 peptide or saidXp-mSDF-1 peptide has a mutation of leucine to proline at the fifthamino acid.
 9. The isolated mutant SDF-1 peptide of claim 6, whereinsaid mutation of the fifth amino acid from the N terminus of said SDF-1peptide is not a mutation to a tryptophan or a glutamic acid amino acidresidue.
 10. The isolated mutant SDF-1 peptide of claim 6, wherein saidisolated mutant peptide is an Xp-mSDF-1 peptide and wherein X is aserine and p equals one.
 11. An isolated mSDF-1 peptide comprising theformula of X_(p)-mSDF-1, wherein said SDF-1 is a peptide comprising theamino acid sequence of at least amino acids 1-8 of SEQ ID NO:52 andwhich is optionally extended at the C terminus by all or any portion ofthe remaining sequence of SEQ ID NO:52, shown as amino acids 9-68, andwherein: a) X is a proteinogenic amino acid or a protease protectiveorganic group; b) p is any integer from 1 to 4; and c) X_(p)-mSDF-1 is amutant form of said SDF-1 comprising a mutation of the fourth, thefifth, or the fourth and the fifth amino acid from the N terminus ofsaid SDF-1, wherein X_(p)-mSDF-1 has chemoattractant activity for Tcells, is inactivated by DPPIV at a rate that is less than one-half ofthe rate at which native SDF-1 is inactivated, and is inactivated byMMP-2 at a rate that is less than one-half of the rate at which nativeSDF-1 is inactivated.
 12. The isolated mutant SDF-1 peptide of claim 11,wherein said Xp-mSDF-1 peptide has a mutation of serine to valine at thefourth amino acid.
 13. The isolated mutant SDF-1 peptide of claim 11,wherein said Xp-mSDF-1 peptide has a mutation of leucine to proline atthe fifth amino acid.
 14. The isolated mutant SDF-1 peptide of claim 11,wherein said mutation of the fifth amino acid from the N terminus ofsaid SDF-1 peptide is not a mutation to a tryptophan or a glutamic acidamino acid residue.
 15. The isolated mSDF-1 peptide of claim 1, 6, or11, wherein said mSDF-1 or said Xp-mSDF-1 peptide maintainschemoattractant activity for T cells of at least 10% that of nativeSDF-1 and is inactivated by MMP-2 at a rate that is less than one-fourthof the rate of inactivation of native SDF-1.
 16. The isolated mutantSDF-1 peptide of claim 11, wherein said isolated mutant peptide is anXp-mSDF-1 peptide and wherein X is a serine and p equals one.
 17. Afusion protein comprising the formula: A-(L)_(n)-(R)_(q), wherein: A isthe isolated mutant SDF-1 peptide of claim 1, 6, or 11; n is an integerfrom 0-3; q is an integer from 1-3; L is a linker sequence of 3-9 aminoacids, and R is a self-assembling peptide selected from the groupconsisting of: AKAKAEAEAKAKAEAE; (SEQ ID NO: 1) AKAEAKAEAKAEAKAE; (SEQID NO: 2) EAKAEAKAEAKAEAKA; (SEQ ID NO: 3) KAEAKAEAKAEAKAEA; (SEQ ID NO:4) AEAKAEAKAEAKAEAK; (SEQ ID NO: 5) ADADARARADADARAR; (SEQ ID NO: 6)ARADARADARADARAD; (SEQ ID NO: 7) DARADARADARADARA; (SEQ ID NO: 8)RADARADARADARADA; (SEQ ID NO: 9) ADARADARADARADAR; (SEQ ID NO: 10)ARADAKAEARADAKAE; (SEQ ID NO: 11) AKAEARADAKAEARAD; (SEQ ID NO: 12)ARAKADAEARAKADAE; (SEQ ID NO: 13) AKARAEADAKARADAE; (SEQ ID NO: 14)AQAQAQAQAQAQAQAQ; (SEQ ID NO: 15) VQVQVQVQVQVQVQVQ; (SEQ ID NO: 16)YQYQYQYQYQYQYQYQ; (SEQ ID NO: 17) HQHQHQHQHQHQHQHQ; (SEQ ID NO: 18)ANANANANANANANAN; (SEQ ID NO: 19) VNVNVNVNVNVNVNVN; (SEQ ID NO: 20)YNYNYNYNYNYNYNYN; (SEQ ID NO: 21) HNHNHNHNHNHNHNHN; (SEQ ID NO: 22)ANAQANAQANAQANAQ; (SEQ ID NO: 23) AQANAQANAQANAQAN; (SEQ ID NO: 24)VNVQVNVQVNVQVNVQ; (SEQ ID NO: 25) VQVNVQVNVQVNVQVN; (SEQ ID NO: 26)YNYQYNYQYNYQYNYQ; (SEQ ID NO: 27) YQYNYQYNYQYNYQYN; (SEQ ID NO: 28)HNHQHNHQHNHQHNHQ; (SEQ ID NO: 29) HQHNHQHNHQHNHQHN; (SEQ ID NO: 30)AKAQADAKAQADAKAQAD; (SEQ ID NO: 31) VKVQVDVKVQVDVKVQVD; (SEQ ID NO: 32)YKYQYDYKYQYDYKYQYD; (SEQ ID NO: 33) HKHQHDHKHQHDHKHQHD; (SEQ ID NO: 34)RARADADARARADADA; (SEQ ID NO: 35) RADARGDARADARGDA; (SEQ ID NO: 36)RAEARAEARAEARAEA; (SEQ ID NO: 37) KADAKADAKADAKADA; (SEQ ID NO: 38)AEAEAHAHAEAEAHAH; (SEQ ID NO: 39) FEFEFKFKFEFEFKFK; (SEQ ID NO: 40)LELELKLKLELELKLK; (SEQ ID NO: 41) AEAEAKAKAEAEAKAK; (SEQ ID NO: 42)AEAEAEAEAKAK; (SEQ ID NO: 43) KAKAKAKAEAEAEAEA; (SEQ ID NO: 44)AEAEAEAEAKAKAKAK; (SEQ ID NO: 45) RARARARADADADADA; (SEQ ID NO: 46)ADADADADARARARAR; (SEQ ID NO: 47) DADADADARARARARA; (SEQ ID NO: 48)HEHEHKHKHEHEHKHK; (SEQ ID NO: 49) VEVEVEVEVEVEVEVEVEVE; (SEQ ID NO: 50)and RFRFRFRFRFRFRFRFRFRF. (SEQ ID NO: 51)


18. The fusion protein of claim 17, wherein the isolated mutant SDF-1peptide comprises a serine to valine mutation at the fourth amino acidor a leucine to proline mutation at the fifth amino acid.
 19. The fusionprotein of claim 17, wherein n=1 and L is selected from the groupconsisting of: GGGGGG (SEQ ID NO:57); GIVGPL (SEQ ID NO:58) and PVGLIG(SEQ ID NO:59).
 20. The fusion protein of claim 17, wherein q=1 and R isRARADADARARADADA (SEQ ID NO:35) or wherein q=1 and R is RADARADARADARADA(SEQ ID NO: 9).
 21. The fusion protein of claim 20, wherein n=1 and L isGGGGGG (SEQ ID NO: 57).